Polymer backbone element tags

ABSTRACT

Element tags based on novel metal-polymer conjugates are provided for elemental analysis of analytes, including ICP-MS. A polymer backbone is functionalized to irreversibly bind metals that are selected prior to use by the user. The polymer is further functionalized to attach a linker which allows for attachment to antibodies or other affinity reagents. The polymer format allows attachment of many copies of a given isotope, which linearly improves sensitivity. The metal-polymer conjugate tags enable multiplexed assay in two formats: bulk assay, where the average biomarker distribution in the sample is diagnostic, and single cell format to distinguish a rare (for example a diseased) cell in a complex sample (for example, blood).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/047,927, filed Jul. 27, 2018, which is a continuation of U.S. patentapplication Ser. No. 14/875,553, filed Oct. 5, 2015, now U.S. Pat. No.10,072,104, which is a divisional of U.S. application Ser. No.14/659,224, filed on Mar. 16, 2015, now U.S. Pat. No. 9,296,838, whichis a divisional application of U.S. application Ser. No. 11/754,340filed on May 28, 2007, now U.S. Pat. No. 9,012,239, which claims thebenefit of U.S. Provisional Patent Application Ser. No. 60/803,356,entitled “Polymer Backbone Elemental Tags,” filed May 27, 2006, theentire contents of which are incorporated by this reference.

COPYRIGHT AND LEGAL NOTICES

A portion of the disclosure of this patent document contains materialwhich is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by anyone of the patent documentor the patent disclosure, as it appears in the Patent and TrademarkOffice patent files or records, but otherwise reserves all copyrightswhatsoever.

FIELD

The invention relates to a new class of tagged biomolecules that havebeen specifically designed to operate in conjunction with elementalanalysis detection, to provide high sensitivity multiplexed biomarkerdeterminations.

INTRODUCTION

Technology that enables accurate protein quantitation is desired in thebooming area of proteomics and drug discovery as well as in areas ofclinical and diagnostic testing. It is also important in biologicalresearch aimed at analyzing protein synthesis, function and disease atthe molecular level. Currently, there are several existing techniquesthat are widely used for estimating protein concentration including:Bradford and Lowry assays, UV spectroscopy, organic mass spectrometry,HPLC, flow cytometry, ligand binding assays, ELISA (Enzyme LinkedImmunosorbent Assay), and RIA (RadioImmunoAssay). Nevertheless, thisextensive assortment of well-established analytical tools and researchtechniques remains insufficient for today's challenges. The failures ofthese methods relate to limitations in sensitivity, selectivity, dynamicrange, and the ability to determine the concentration of severalproteins simultaneously in an accurate and absolute manner(multiplexing). The realization that elemental analysis offerssignificant advantages to the field of protein quantitation has directedthe development of several new methods of protein quantitation viaInductively Coupled Plasma Mass Spectrometry (ICP-MS) linkedimmunoassays¹⁻⁴. This new technique provides an innovative arena forICP-MS in the analysis of biological samples^(5;6). The uniqueanalytical properties of ICP-MS allow the selection of tags from thenon-radioactive elements that do not naturally occur in biologicalsamples.

Definitions

“Elemental analysis” is a process where a sample is analyzed for itselemental composition and/or isotopic composition. Elemental analysiscan be accomplished by a number of methods, including, but not limitedto:

-   (i) optical atomic spectroscopy, such as flame atomic absorption,    graphite furnace atomic absorption, and inductively coupled plasma    atomic emission, which probe the outer electronic structure of    atoms;-   (ii) mass spectrometric atomic spectroscopy, such as inductively    coupled mass spectrometry, which probes the mass of atoms;-   (iii) X-ray fluorescence, particle induced x-ray emission, x-ray    photoelectron spectroscopy, and Auger electron spectroscopy which    probes the inner electronic structure of atoms.

“Elemental analyzer” is an instrument for the quantitation of the atomiccomposition of a sample employing one of the methods of elementalanalysis.

“Particle elemental analysis” is a process where a sample, composed ofparticles dispersed in a liquid (beads in buffer, or cells in growthmedia, or blood, for example), is interrogated in such manner that theatomic composition is recorded for individual particles (bead-by-bead,cell-by-cell, particle-by-particle, for example). An example of theanalytical instrument is a mass spectrometer-based flow cytometer,ICP-TOF, or ICP-MS or any elemental analyzer configured to interrogateindividual particles.

“Volume or bulk elemental analysis” is a process where an analyzedsample is interrogated in such manner that the atomic composition isaveraged over the entire volume of the sample.

“An internal standard” is defined as a known amount of a compound,different from the analyte that is added to the unknown. Signal fromanalyte is compared with signal from the internal standard to find outhow much analyte is present. An internal standard may be used whenperforming mass spectrometry quantitation. An internal standard can bealso used by other means known to those skilled in the art.

“Biological sample” refers to any sample of a biological nature thatrequires analysis. For example, the sample may comprise or may besuspected of comprising biological molecules, tissue, fluid, and cellsof an animal, plant, fungus, or bacteria. It also includes molecules ofviral origin. Typical samples include, but are not limited to, sputum,blood, blood cells (e.g., white cells), tissue or fine needle biopsysamples, urine, peritoneal fluid, and pleural fluid, or cells therefrom.Biological samples may also include sections of tissues such as frozensections taken for histological purposes. Another typical source ofbiological samples are viruses and cell cultures of animal, plant,bacteria, fungi where gene expression states can be manipulated toexplore the relationship among genes. Biological samples may alsoinclude solutions of biological molecules (either purified or notpurified) such as proteins, peptides, antibodies, DNA, RNA, aptamers,polysaccharides, lipids, etc. Other examples are known to those skilledin the art.

“Antibodies” are immunoglobulin glycoprotein molecules found normally inserum of animals. Antibodies may be made in mammals such as rabbits,mice, rats, goats, etc., and chicken or may be made by recombinantmethods as is known to those skilled in the art and described, forexample, in U.S. Pat. No. 4,816,567. Procedures for immunization andelicitation of a high antibody production response in an animal are wellknown to those skilled in the art and can be found, for example, inAntibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988,pages 92-115. Antibodies may be used as whole molecules, fragments ofmolecules known as Fab′ and Fab2′ fragments, as monovalent antibodies(combining a light chain and a modified heavy chain), and other examplesknown in to those skilled in the art.

“Primary antibodies” are antibodies that bind specifically to an antigeninjected into an animal. They can be extracted from the animal or can bemade by recombinant means.

“Secondary antibodies” are those antibodies that bind specifically toprimary antibodies. For example, primary antibodies can be used as theantigen injected into an animal of a different species, to generatesecondary antibodies. For example, rabbit secondary anti-mouseantibodies can be made by immunizing a rabbit with mouse antibodies.

“Antigen” is a substance that stimulates an immune response in a hostorganism, especially the production of antibodies. Antigens are usuallyproteins or polysaccharides, but can be any type of molecule, includingbut not limited to, small molecules (haptens) coupled to acarrier-protein.

“Bio-markers” are molecules and constructs, which may for example beantigens, small molecules, nucleotides, oligonucleotides, DNA or RNA,that are present in the cell volume or on the cell surface of only onetype of cell, or whose relative abundance is unique to that type ofcell. Cell bio-markers can be used to distinguish that cell from othercells. For example, antigens present on the cell surface of only onetype of cell are called cell surface bio-markers that distinguish thatcell from other cells.

“Immunoassay” as used herein means an assay in which an analyte, such ascellular antigen or bio-marker, is detected by an affinity reagent suchas a primary antibody. For example, an “immunoassay” can be an assay inwhich an analyte is detected by a tagged affinity reagent, such as aprimary antibody conjugated to a metal tagged polymer.

“Biomolecule” as used herein means any biological molecule and includessmall biomolecules, for example, but not limited to: Lipids,Phospholipids, Glycolipids, Sterols, Vitamins, Hormones,Neurotransmitters, Carbohydrates, Sugars, Disaccharides, Amino acids,Nucleotides, Phosphate, and Monosaccharides; and large biomolecules, forexample but not limited to: Peptides, Oligopeptides, Polypeptides,Proteins, Nucleic acids, i.e. DNA, RNA, Oligosaccharides,Polysaccharides, and Prions. Other biomolecules are known to thoseskilled in the art and are encompassed in the applicant's teachings.

“Affinity reagent” is a biomolecule capable of tightly binding to atarget molecule or analyte, for example an antigen or biomarker. Forexample, an antibody is an affinity reagent that recognizes and bindswith high affinity to a specific antigen. Streptavidin is a proteinmolecule that specifically binds biotin and may be considered as anotherexample of the affinity reagent. Other affinity reagents are known tothose skilled in the art, and include, but are not limited to aptamers,oligonucleotides, protein molecules, lectins and polysaccharides.

“Tagged affinity reagent” is an affinity reagent (for example, anantibody or aptamer or oligonucleotide, polysaccharides, lipids,hormones, growth factors) that is conjugated to a synthetic tag (moiety)usually through a linker group. The tag can be, but is not limited to, apolymer with covalently attached multiple chelating groups. To a greaterextent, the chelating groups can have an element or multitude ofelements attached to them. The sequence and order of the chelation stagedepends on the tagging protocol.

The term “detect” is used in the broadest sense meaning to include bothqualitative and quantitative measurements of a specific molecule. Forexample, qualitative and quantitative measurements of a specific antigenor biomarker with the help of a tag (for example, a tagged antibody orother tagged affinity reagent).

“Element tag” or “tag” is a chemical moiety which includes an element ormultitude of elements having one or many isotopes (referred to as “tagatoms”) attached to a supporting molecular structure, or that is capableof binding said element(s) or isotope(s). The element tag can alsocomprise the means of attaching the element tag to a molecule ofinterest or target molecule (for example, an analyte). Different elementtags may be distinguished on the basis of the elemental composition ofthe tags. An element tag can contain many copies of a given isotope andcan have a reproducible copy number of each isotope in each tag. Anelement tag is functionally distinguishable from a multitude of otherelement tags in the same sample because its elemental or isotopiccomposition is different from that of the other tags.

The term “tag atom” is the atom of the element or isotope thatdifferentiates one element tag from another and that is detected byelemental analysis.

“A support” is a surface which has been functionalized by, for example,pyrrole-2,5-dione (maleimido), sulfonic acid anion, or p-(chloromethyl)styrene. A support, for example, may be but is not limited to, asynthetic membrane, bead (polystyrene, agarose, silica, etc), planarsurface in plastic microwells, glass slides, reaction tubes, etc. as isknown to those skilled in the art.

“ICP-MS” is the Inductively Coupled Plasma Mass Spectrometer—a sensitivemass spectrometry based elemental analyzer. Different ICP-MSconfigurations are primarily distinguished by the mass selectingtechnique employed and can be, for example the quadrupole ortime-of-flight (ICP-TOF) or magnetic sector (high resolution ICP-MS).There are many commercially available ICP-MS models having a widespectrum of configurations, capabilities and modifications.

A “polymer” is a substance composed of molecules characterized by themultiple repetitions of one or more species of atoms or groups of atoms(constitutional units) linked to each other in amounts sufficient toprovide a set of properties that do not vary markedly with the additionor removal of one or a few constitutional units. (IUPAC definition, seeE. S. White, J. Chem. Inf. Comput. Sci. 1997, 37, 171-192). A polymermolecule can be thought of in terms of its backbone, the connected linkof atoms that span the length of the molecule, and the pendant groups,attached to the backbone portion of each constituent unit. The pendantgroups are often chemically and functionally different from the backbonechain. Pendant groups that have a high affinity for metal ions can actas chelating groups or ligands for those ions.

“Copolymers” are polymers that consist of two or more chemicallydifferent constituent units. A “linear polymer” is a polymercharacterized by a linear sequence of constituent units. A “blockcopolymer” is a linear polymer with sequences of constituent units of acommon type, joined to sequences of constituent units of a differenttype. A “branched polymer” is a polymer in which additional polymerchains (the branches) issue from the backbone of the polymer. Onecommonly refers to the longest linear sequence as the “main chain”. Abranched polymer in which the chemical composition of the constituentunits of the branch chains is different than those of the main chain iscalled a “graft copolymer”.

“Star polymers” have multiple linear polymer chains emanating from acommon constituent unit or core. “Hyperbranched polymers” are multiplebranched polymers in which the backbone atoms are arranged in the shapeof a tree. These polymers are related to “dendrimers”, which have threedistinguishing architectural features: an initiator core, interiorlayers (generations) composed of repeating units radially attached tothe initiator core, and an exterior surface of terminal functionalityattached to the outermost generation. “Dendrimers” differ fromhyperbranched polymers by their extraordinary symmetry, high branching,and maximized (telechelic) terminal functionality.

A “metal tagged polymer” (also a “polymeric metal tag carrier”, or“metal-polymer conjugate”, or “chelate-derivatized polymer”) is avariety of the element tag which consists of a polymer backbone bearingat least one pendant chelating group with metal atoms attached to them.These metal tagged polymers can be, but are not limited to, linear,star, branched, or hyperbranched homopolymers or copolymers as well asblock or graft copolymers.

A “metal binding pendant group” is a pendant group on the polymer thatis capable of binding a metal or an isotope of a metal. It can also bereferred to as a ligand.

An “attachment (linker) group” or “linker” is a portion of a moleculethat is used to couple (conjugate) two different molecules or polymers,two subunits of a molecule, or a molecule to a substrate, for example anaffinity agent.

Commonly used abbreviations: NAS is N-acryloxysuccinimide; NMAS isN-methacryloxysuccinimide; DMA is N,N-dimethylacrylamide; t-BDB is thereversible addition-fragmentation chain transfer (RAFT) chain transferagent, tert-butyl dithiobenzoate; AMBN is 2,2-azobis(2-methylbutyronitrile); DMSO is Dimethyl Sulfoxide; DOTA is1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; PMAA ispoly(methacrylic acid); DTPA is diethylenetriamine pentaacetic acid;PDMAEMA is poly(dimethylaminoethyl methacrylate); Fmoc is9-fluorenylmethyl carbamate; DTT is dithiothreitol; TMS istrimethylsilyl and TCEP is tri(2-carboxyethyl)phosphine.

The terms Mn, Mw and PDI (polydispersity index): Mw/Mn are used toindicate the number and average molecular weight and the polydispersityindex describes the molecular weight distribution, respectively.

“Chelation” is the process of binding of a ligand, the chelant, chelatoror chelating agent, to a metal ion, forming a metal complex, thechelate. In contrast to the simple monodentate ligands like H₂O or NH₃,the polydentate chelators form multiple bonds with the metal ion.

“Transition element” means an element having one of the following atomicnumbers 21-30, 39-48, 57-80 and 89-92. Transition elements include therare earth metals, lanthanides and noble metals.

“Lanthanides” are the transition metals with atomic numbers from 57 to71 including La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu.

“Metal” means an element having one of the following atomic numbers 3,4, 11-13, 19-33, 37-52, 55-84, 87-102.

SUMMARY

A class of tags optimized for elemental analysis including (but limitedto) the ICP-MS application has not before been developed. Preliminarystudies had to be done^(1;3;4;7) using tags that are currently in usefor completely different purposes. The element tags of the presentinvention are not those of the prior art and are specifically designedfor elemental analysis. To implement elemental tagging to its fullest,the development of a new class of tags was required. Inductively CoupledPlasma Mass Spectrometry (ICP-MS) has a number of unique properties thatcan be harnessed to create an ideal elemental tag instrumentcombination. The most important advantage is the fact that a largenumber of heavy metals and their isotopes provide distinct signals thatcan be detected simultaneously. Thus many, for example greater than 50,element tags can be developed; the obtained intensity of tag elementsserves as a signature of the analyte concentration in the sample.Secondly, the abundance sensitivity of ICP-MS, a measure of the overlapof signals of neighboring isotopes, is large (for example greater than10⁶ for the quadrupole analyzer), and this ensures independence of thedetection channels over a wide dynamic range. The third key property isthat MS is very sensitive; detection on the order of 100 molecules of agiven antigen per cell may be feasible, and largely independent of theorder of multiplex, a substantial improvement over current fluorescencecytometer instruments. Finally, ICP-MS as a detector offers absolutequantification that is largely independent of the analyte molecular formor sample matrix. There is a definite need to integrate these keyproperties of elemental analysis with bio-analytical methodology. Here,we provide a novel design of the element tags, which ensuresdramatically higher multiplex capability and sensitivity of bio-assays.

The new class of polymer based element tags is suitable fordetermination using conventional ICP-MS instruments in the instance thatan average assay over a sample ensemble (i.e., bulk assay) is desired.For example, where a tissue is sufficiently homogeneous, or thediagnostic allows for averaging over the biopsy, the sample may bestained with the metal-tagged affinity reagents and, following washing,may be acidified to lyse the cells of the tissue and provide ahomogeneous solution that can be analyzed according to long-standingstandard ICP-MS protocols. The bulk assay protocol still allows formassively multiplexed assay, with detection limits for each markercomparable to individual radio-immunoassay. Cell biologists might viewthis as a quantitative high-throughput analog of Western blotting.

The new class of polymer based element tags is suitable fordetermination using a novel flowcytometry ICP-MS based instrument⁸ andprovide up to 50 or more distinguishable reporter tags for immunologicalassays that enable the simultaneous determination (massivelymultiplexed) of many biomarkers, ultimately providing exquisitedistinction and identification of diseased cells (or other cells ofinterest) in patient's samples in particle elemental analysis.

The new class of polymer based element tags is suitable for doublelabeling of affinity reagents—fluorescent label and element tag on thesame affinity reagent. Previously, double labeled antibodies were usedto localize specific cell types in tissue sections (fluorescentmicroscopy) and then identify the particular structures of cells usingelectron microscopy. Therefore, antibodies were labeled withfluoresceneisothiocyonate (FITC) and ferritin as an electron densematerial.⁹ More recently, immunoprobes that combine a fluorescent labelwith a small gold cluster have been prepared by covalent conjugationwith Fab′ fragments. These new immunoconjugates allow the collection oftwo complementary sets of data, from fluorescence and electronmicroscopy, from a single labeling experiment.¹⁰ Another advance inreagents such as terbium-fluorescein and terbium-green fluorescentprotein fluorescence resonance energy transfer pairs was achieved tostudy kinase reactions using Time Resolved-Fluorescence Resonance EnergyTransfer (TR-FRET)¹¹.

The Applicant's teaching includes double labeled affinity reagents tofacilitate presorting and subsequent elemental analysis of rare cells inmixed samples by ICP-MS-based flow cytometry. Cell biology requiresmicroscopic localization of biomarkers on the cell surface orintracellularly. At the same time, quantitative information on theabundance of the markers is necessary. By covalently attaching afluorescent label and an element tag to the same affinity reagent (forexample an antibody) and using this affinity reagent, first, to localizethe signal to a particular subcellular structure (membrane, nucleus,cytoplasm, cytoskeleton, etc) via fluorescent microscopy and, second, toquantify the number of bound affinity reagents by ICP-MS, willsignificantly increase biological understanding of processes underinvestigation.

An aspect of the invention is to provide an element tag comprising apolymer, wherein the polymer comprises at least one metal-bindingpendant group that comprises at least one metal atom or is capable ofbinding at least one metal atom. The element tag can further comprise afunctional group that allows the polymer to be attached to one of alinker, a spacer, or a biomolecule. The element tag can be watersoluble. It can also be negatively charged. The number of metal-bindingpendant groups capable of binding at least one metal atom can be betweenapproximately 1 and 1000, and most typically between approximately 10and 250. At least one metal atom can be bound to at least one of themetal-binding pendant groups. The polymer can have a degree ofpolymerization of between approximately 1 and 1000, and most typicallybetween 10 and 250.

The polymer can be selected from the group consisting of linearpolymers, copolymers, branched polymers, graft copolymers, blockpolymers, star polymers, and hyperbranched polymers. The backbone of thepolymer can be derived from substituted polyacrylamide,polymethacrylate, or polymethacrylamide and can be a substitutedderivative of a homopolymer or copolymer of acrylamides,methacrylamides, acrylate esters, methacrylate esters, acrylic acid ormethacrylic acid.

The metal-binding pendant group can be attached to the polymer throughan ester or through an amide. The functional group can be athiol-reactive group. The metal atom can be a transition element or anisotope thereof, or a lanthanide or an isotope of a lanthanide. Theelement tag can further comprise a linker attached to the functionalgroup of the polymer, wherein the linker is capable of covalentattachment to a biomolecule. The element tag can further comprise aspacer attached to the linker, wherein the spacer is capable ofattachment to a biomolecule. The spacer can be a polyethylene glycol(PEG) spacer. The spacer can comprise a functional group that is capableof binding the spacer to the polymer via a spacer-reactive functionalgroup on the polymer. Further the spacer can contain a functional groupthat is capable of binding a linker to the spacer.

The element tag described above, can be covalently attached to abiomolecule. The biomolecule can be an affinity reagent, and theaffinity reagent can be an antibody.

Another aspect of the invention is to provide an element tagged affinityreagent, wherein the affinity reagent is tagged with the element tagdescribed above, and wherein at least one of the pendant groups binds,or is capable of binding, at least one metal atom.

Another aspect of the invention is to provide a method of preparing theelement tag described above, comprising: (i) providing a polymer; and(ii) covalently attaching at least one metal-binding pendant groupcontaining at least one metal atom or capable of binding at least onemetal atom to the polymer. The step of providing the polymer cancomprise synthesis of the polymer wherein the synthesis is selected fromthe group consisting of reversible addition fragmentation polymerization(RAFT), atom transfer radical polymerization (ATRP) and anionicpolymerization. The step of providing the polymer can comprise synthesisof the polymer from compounds selected from the group consisting ofN-alkyl acrylamides, N,N-dialkyl acrylamides, N-aryl acrylamides,N-alkyl methacrylamides, N,N-dialkyl methacrylamides, N-arylmethacrylamides, methacrylate esters, acrylate esters and functionalequivalents thereof. The metal-binding pendant group that is capable ofbinding at least one metal atom can comprise adiethylenetriaminepentaacetate (DTPA) ligand or a1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) ligand.The method can further comprise functionalizing the polymer, wherein thefunctional group is capable of covalently binding a biomolecule. Themethod can further comprise attaching a linker to the functional groupof the polymer, wherein the linker is capable of binding a biomolecule.The method can further comprise covalently binding a biomolecule to thelinker. Finally, the method can further comprise binding at least onemetal atom to at least one metal-binding pendant group.

Another aspect of the invention is to provide a method of preparing theelement tag described above, comprising: (i) providing a polymercomprising at least one metal-binding pendant group that contains atleast one metal atom or is capable of binding at least one metal atom,and comprising a functional group that allows the polymer to covalentlybind a linker; (ii) attaching a linker to the functional group of thepolymer, wherein the linker is capable of binding a biomolecule; (iii)covalently binding a biomolecule to the linker; and (iv) binding atleast one metal atom to at least one metal-binding pendant group. Thestep of binding at least one metal atom to at least one metal-bindingpendant group can be performed before step (ii). The step of binding atleast one metal atom to at least one metal-binding pendant group can beperformed before step (iii). The step of binding at least one metal atomto at least one metal-binding pendant group can be performed after step(iii). The method can further comprise a step of attaching a spacer tothe linker, wherein the spacer lies between the linker and thebiomolecule and/or a step of attaching a spacer to the polymer, whereinthe spacer lies between the polymer and the linker. The spacer can beadded before step (ii). The spacer can be a polyethylene glycol (PEG)spacer. The spacer can contain a functional group that is capable ofbinding the spacer to the polymer via a spacer-reactive functional groupon the polymer. The spacer can contain a functional group that iscapable of binding the spacer to the linker. The method can include astep of reacting the thiol with a maleimido attachment group.

Another aspect of the invention is to provide an element tag prepared bythe methods described above.

Another aspect of the invention is a method for the analysis of ananalyte, comprising (i) incubating the element tagged affinity reagentdescribed above with an analyte, wherein the affinity reagent binds withthe analyte; (ii) separating unbound tagged affinity reagent from boundaffinity reagent; and (iii) analyzing the element bound to the affinityreagent attached to the analyte by elemental analysis.

Another aspect of the invention is to provide a method for the multiplexanalysis of two or more analytes, comprising: (i) incubating two or moredifferential element tagged affinity reagents described above with twoor more analytes, wherein the affinity reagents bind with the analytes,to produce two or more differentially tagged analytes; (ii) separatingunbound affinity reagents from bound affinity reagents; and (iii)analyzing the differential tags bound to the two or more analytes byelemental analysis.

Another aspect of the invention is to provide a method for the analysisof an analyte, comprising: (i) incubating the element tag describedabove with an analyte, so that the element tag binds the analyte; (ii)separating unbound tag elements from bound tag elements; and (iii)analyzing the bound tag elements by elemental analysis.

The affinity reagent of any of the above methods can further labeledwith a fluorescent label. The analyte can be located within or on acell, for example a diseased cell, and further a leukemia cell. The stepof analysis can comprise bulk analysis, wherein the atomic compositionis averaged over an entire volume of a sample, and/or analysis of singleparticles. The particles can be cells.

The methods described above can be done by elemental analysis by ICP-MSor by a mass spectrometer based flow cytometer.

Another aspect of the invention is to provide a kit for the preparationof the element tag described above, comprising at least one of thefollowing: a polymer comprising at least one metal-binding pendant groupwhich comprises at least one metal atom or is capable of binding atleast one metal atom and further comprising a functional group thatallows the polymer to be attached to one of a linker, a spacer, or abiomolecule, a metal solution, reagents for the attachment of thelinker, spacer or biomolecule to the polymer, reagents for attachment ofa functional group to the linker or the spacer, reagents for attachmentof a metal to the polymer, affinity reagents including antibodies,buffers, instructions for preparing the element tag, instructions forattaching the element tag to an affinity reagent, and instructions forattaching a metal to the element tag.

Another aspect of the invention is to provide a kit for the analysis ofanalytes according to the methods described above comprising at leastone of the following: a polymer comprising at least one metal-bindingpendant group which contains at least one metal atom or is capable ofbinding at least one metal atom and further comprising a functionalgroup that allows the polymer to be attached to one of a linker, aspacer, or a biomolecule, a metal solution, reagents for the attachmentof the linker, spacer or biomolecule to the polymer, reagents forattachment of a functional group to the linker or the spacer, reagentsfor attachment of a metal to the polymer, affinity reagents includingantibodies, buffers, instructions for preparing the element tag,instructions for attaching the element tag to an affinity reagent,instructions for attaching a metal to the element tag. and instructionsfor using the element tags for the analysis of analytes by elementalanalysis.

The polymer for any of the above kits can be selected from the groupconsisting of homopolymers or copolymers of acrylamides,methacrylamides, acrylate esters, methacrylate esters, acrylic acid andmethacrylic acid. The reagents can include at least one of thefollowing: TCEP (tri(2-carboxyethyl)phosphine),Ligand-Polymer-Linker-Spacer Conjugate, phosphate buffer, TBS(tris-buffered saline), EDTA (Diaminoethanetetraacetic acid), ammoniumacetate buffer, antibodies, metal salt solution, lanthanide saltsolution, blocker buffers, washing buffers, FBS (fetal bovine serum),DMEM (Dulbecco's Modified Eagle's Medium), BSA (bovine serum albumin),dithiothreitol, bismaleimide, and DMF (dimethylformamide). The polymercan be attached to a linker or it can be attached to a linker and aspacer.

These and other features of the applicant's teachings are set forthherein.

BRIEF DESCRIPTION OF THE FIGURES

The invention is illustrated in the figures of the accompanyingdrawings, which are meant to be exemplary and not limiting, and in whichlike references are intended to refer to like or corresponding parts.

FIG. 1. Schematic views of the element tags for the detection ofbiomolecules which according to the invention have the general structureI. Proposed polymeric metal chelates: R=organic group, L=Metal ligand.In structure “a” each repeat unit of the polymer bears the ligandedLn3+, denoted by (L). In structure “b”, a fraction of the repeat unitshave an organic group R according to the invention. Asterisk (*)represents the initiated end of the polymer NAS is schematic view ofN-acryloxysuccinimide. NMAS is schematic view ofN-methacryloxysuccinimide.

FIG. 2. Schematic views of an example of the synthesis of functionalligands that can be used to attach the element/metal “L” to the polymer.

FIG. 3. Schematic views of attaching ligands (pending groups) to theRAFT polymers (Scheme 3) and ATRP polymers (Scheme 4).

FIG. 4. Schematic views of attaching ligands (pending groups) topolymers produced by anionic polymerization (Scheme 5) and PDMAEMA(Scheme 6).

FIG. 5. Schematic views of attachment of the coupling group (the linker)to the RAFT polymers (Scheme 7a), ATRP polymers (Scheme 7b), andpolymers produced by anionic polymerization (Scheme 7c).

FIG. 6. Schematic views of alternative examples of coupling chemistryaccording to the invention. In scheme 8b, the term “end-group” is usedto refer to the coupling group.

FIG. 7. Schematic views of structures of monomers.

FIG. 8. Experimental conditions and molecular weight data for randomcopolymers of DMA and NAS in dioxane at 80° C.

FIG. 9. Schematic views of preparation of ligand-polymer conjugate.

FIG. 10. Schematic views of preparation of the DOTA based ligand-polymerconjugate.

FIG. 11. Schematic views of synthesis of the element tag.

FIG. 12. Schematic views a process to generate a polymer with pendentamino groups for attachment of DTPA ligands and of employing a newinitiator based on cystamine.

FIG. 13. Is a bar graph of the results of Experiment 6. Growing K562cells (non-differentiated) were stained with primary antibodies labeledwith Ligand-Polymer Conjugate (as described in Scheme 11)—carryingidentifying lanthanides: anti-CD38 monoclonal antibody was labeled withLa; anti-CD110—with Eu; anti-CD61—with Dy; anti-CD45—with Ho;anti-CD54—with Nd; CD49d—with Pr. Cells were reacted with labeledantibodies either with each separately, or with all antibodiessimultaneously (ALL; 6-plexing). Note that the highly expressedubiquitous nucleated blood cell marker CD45 (Ho) is on average 10 timesgreater than cell adhesion markers (CD54, Cd38, CD49d), and 100 timesgreater than megakaryocite differentiation markers CD61 and CD110 (cellswere not induced to differentiate along the megakaryocite pathway).

FIG. 14. a. Is a three-dimensional bar graph showing the directcomparison of fluorescence obtained from cells stained with CD33-FITC ordual labeled CD33-FITC-Pr using flow cytometry. b. Is athree-dimensional bar graph showing the direct comparison of normalizedresponse obtained from cells stained with CD33-Pr or dual labeledCD33-FITC-Pr using ICP-MS.

FIG. 15 is a flow Chart I of RAFT polymerization procedure.

FIG. 16 is a flow Chart II of Polymer-DTPA-Linker attachment procedure.

DESCRIPTION OF THE VARIOUS EMBODIMENTS

The overall requirements for an element tag are less stringent thanthose for a fluorescent tag¹² since the chemical nature of an element isnot important for its detection by elemental analysis. The tag shouldcontain a reproducible and, preferably, large number of atoms of a givenelement or isotope composition. The tag can comprise one element orisotope, or consist of a composition of more than one element orisotope. It can also include a natural mixture of isotopes. Further, itis possible that the element tag can comprise one pendant groupcomprising a certain metal or isotope and a second pendant groupcomprising another metal or isotope. Reproducibility in the number ofidentical atoms incorporated is a basis for quantitative analysis, andan increase in the number of those atoms improves the sensitivitylinearly. Another key attribute is resistance to leaching, whichdistinguishes this invention from the DELFIA products. Mobility of thechelated metal is required in the DELFIA products (DELFIA® Assays andReagents, PerkinElmer, USA). The tag atoms can be any atoms of anelement or isotope that differentiate the tag from other atoms in thesample including from other tag atoms associated with differentiatedelement tags. Typically, the tag atoms will be metals, in particulartransition elements, and most typically lanthanides.

The tags to be employed for the detection of analytes have the generalstructure I of FIG. 1.

The polymer can be any polymer as is known to those skilled in the art.Examples of polymers are shown in FIGS. 1 through 4. Further, thepolymer backbone can be derived from a substituted polyacrylamide,polymethacrylate, or polymethacrylamide. Further still, the backbone ofthe polymer can be a substituted derivative of a homopolymer orcopolymer of acrylamides, methacrylamides, acrylate esters, methacrylateesters, acrylic acid or methacrylic acid. The polymer can be synthesizedby many methods as are known to those skilled in the art. For example,the synthesis can be accomplished with compounds such as N-alkylacrylamides, N,N-dialkyl acrylamides, N-aryl acrylamides, N-alkylmethacrylamides, N,N-dialkyl methacrylamides, N-aryl methacrylamides,methacrylate esters, acrylate esters and functional equivalents thereof.

The ligand or pendant group can be any ligand as is known to thoseskilled in the art. Examples of ligands are shown in FIGS. 2 through 4.

The linker can be any linker as is known to those skilled in the art.Examples of linkers are shown in FIGS. 5 and 6. The linker is optional.

The spacer is optional. Examples of spacers include PEG block spacers,and others known to those skilled in the art.

The invention involves primarily but not exclusively the followingaspects:

(i) Polymeric metal tag carrier synthesis. Functionally, the metaltagged polymer is stable under typical assay conditions, which includesvery low kinetic lability of bound metals and rate of exchange of metalsbetween polymers;

(ii) Synthesis and characterization of the attachment (linker) group incombination with polymeric metal tag carrier;

(iii) Synthesis of tagged affinity reagent, which functionally includesan attachment (linker) group in combination with the polymeric metal tagcarrier. The tagged affinity reagent can be a tagged antibody or othertagged affinity reagent; and

(iv) Method of employing the affinity reagents as multiplexing tools.

More generally the invention involves synthesis and testing ofmetal-containing tags for labeling of bio-organic molecules, includingaffinity reagents such as antibodies. Specifically designed forelemental analysis, such a tag would typically be: (i) water soluble,(ii) non-toxic, (iii) easily separated from a tagged material by knownchromatographic, centrifugation, filtration or dialysis methods; and, inaddition, can have three or four moieties: the attachment group(linker), possibly a spacer (for example, a PEG spacer), the polymerskeleton (carrier), and the tag atoms (as many tag atoms (of the samemetal or isotope, or of a different metal and/or isotope) as possible).For different elemental analyzers the characteristics of the element tagcan be similar.

Although an embodiment of the invention using antibodies as the affinityreagent is exemplified, it is to be understood that other affinityreagents can be used and are within the scope of the invention.

Polymer carrier: An important aspect of the invention is the synthesisof a polymer, to which a large number of tag atoms can be attached.Typically the tag atoms are metal atoms. The polymer can be watersoluble This moiety is not limited by chemical content. However, itsimplifies analysis if the skeleton has a relatively reproducible size(for example, length, number of tag atoms, reproducible dendrimercharacter, etc.). The requirements for stability, solubility, andnon-toxicity are also taken into consideration. Thus, the preparationand characterization of a functional water-soluble polymer by asynthetic strategy that places many functional groups along the backboneplus a different group at one end that can be used to attach the polymervia a linker to a biomolecule (for example, an affinity reagent) is partof this invention.

The tags to be employed for the detection of analytes have the generalstructure I of FIG. 1. The signal to be detected will be that of thepolymer, which will contain between approximately 1 to 1000 (or more)atoms of an element (for example, lanthanide (Ln) atoms) as part of itsstructure. A flexible linker/spacer at one end of the polymer maycontain a thiol-reactive functional group such as a maleimide, andthrough this group can be linked to an affinity reagent (for example anantibody) for the specific target analyte. Variations include theattachment to primary amines of biomolecules or other methods ofattachment known to persons skilled in the art. Examples of theselection of functional groups for the linker arm can be taken from theliterature on PEGylated antibodies, reviewed recently by Chapman¹³. Thepolymers as carriers of the metal-atom tags have a similar number ofbackbone atoms as those of the PEG polymers that have been attached tovarious antibodies without loss of binding affinities. For example aPEG2000 (2 KDa) has a mean degree of polymerization of 45 correspondingto 140 backbone atoms, and PEG5000 has 340 backbone atoms. To put thesetags in perspective, the average size of an IgG antibody from the end ofthe Fc to the Fab is approximately 11 nm¹⁴. The radius of gyration ofthe polymer constructs should be as small as possible, somewhere betweenapproximately 2 nM and 11 nM.

In one embodiment, the invention involves, polymers containing the Ln3+atoms as substituents of the pendant groups and their synthesis. Instructure “a” of FIG. 1, each repeat unit of the polymer bears theliganded Ln3+, the group being denoted by (L). It is neither likely norrequired that each pendant group bear an (L) substituent. In structure bof FIG. 1, a fraction of the repeat units have an organic group R. Inthese structures, the asterisk (*) represents the initiated end of thepolymer. The following factors are considered: 1) The polymer can bewater soluble. Because of their hydrolytic stability, N-alkylacrylamides, N-alkyl methacrylamides, and methacrylate esters orfunctional equivalents can be used. 2) A degree of polymerization (DP)of approximately 1 to 1000 (1 to 2000 backbone atoms) encompasses mostof the polymers of interest. Larger polymers are in the scope of theinvention with the same functionality and are possible as would beunderstood by practitioners skilled in the art. Typically the degree ofpolymerization will be between 10 and 250. 3) The polymers may beamenable to synthesis by a route that leads to a relatively narrowpolydispersity. The polymer may be synthesized by atom transfer radicalpolymerization (ATRP) or reversible addition-fragmentation (RAFT)polymerization, which should lead to values of Mw/Mn in the range of 1.1to 1.2. An alternative strategy involving anionic polymerization, wherepolymers with Mw/Mn of approximately 1.02 to 1.05 are obtainable. Bothmethods permit control over end groups, through a choice of initiatingor terminating agents. This allows synthesizing polymers to which thelinker can be attached. 4) A strategy of preparing polymers containingfunctional pendant groups in the repeat unit to which the ligandedtransition metal unit (for example a Ln unit) can be attached in a laterstep can be adopted. This embodiment has several advantages. It avoidscomplications that might arise from carrying out polymerizations ofligand-containing monomers. In addition, the polymer backbone is a knownone that can be adapted for most if not all of the Ln-containingpolymers. Thus the polymers may have a common mean chain length andchain-length distribution. 5) The target polymers of type “a” may eitherbe negatively charged polyelectrolytes or have zwitterionic pendantgroups. To minimize charge repulsion between pendant groups, the targetligands for (Ln3+) should confer a net charge of −1 on the chelate. Fortype “b” polymers, the R groups are for the most part uncharged,although in one example, the inventors teach a polymer in which thesmall fraction x of R groups will have a positive charge. Finally,various chemistries are well known that enable the attachment of thelinker group with its thiol reactive group to the polymer. A number ofpendant groups can be added to the polymer. Practically, the number canbe between 1 and 1000, and more typically between 10 and 250. Themetal-binding pendant group can be attached to the polymer by methodsknown to those skilled in the art, for example, the pendant group may beattached through an ester or through an amide.

Examples for the synthesis of functional ligands that are used to attach(L) to the polymer are shown in FIG. 2 (Schemes 1 and 2). The examplesare exemplary and are not intended to limit the scope of the invention.

Chelate (tag atom) choice and synthesis: The use of the lanthanides isestablished here as feasible, however, similar results can be achievedfor different elements. Across the series of lanthanides very similarcoordination chemistry is observed. All the lanthanides favor the +3oxidation state and preferentially coordinate with hard oxygen ligands.Lanthanides do not exhibit defined coordination geometries and vary incoordination number between 7 and 10 across the series. Thus, the samechelate-derivatized polymer can be used for all the Ln metals, whichfacilitates production of tags containing different lanthanides used inmultiplexing assays¹⁵. Different embodiments utilizing different metalscan be obtained using similar considerations related to their chemicalnature. Numerous Ln complexes have been developed for use asradiopharmaceuticals and imaging agents¹⁶. But the art does not disclosemetal atoms attached to pendant groups on the polymer backbone. Themultidentate chelates developed for these applications formthermodynamically stable and kinetically inert Ln complexes, importantfor minimizing the toxicity of free lanthanides in vivo. Incorporatingthese optimized lanthanide chelates, as pendant groups on polymericstructures, appears to be described here for the first time.

As examples, two ligand frameworks as functional examples of covalentlylinked chelates on the polymeric backbone are described. The selectioncriteria for this embodiment include known syntheses, heptadentate oroctadentate coordination to promote kinetic stability against metal iondissociation, a pendant primary amine functional group for attachment ofthe chelate to the polymer, and a net charge of −1 for the ligandedchelate. Diethylenetriaminepentaacetate (DTPA), an acyclic chelator canbe readily derivatized as an amine functionalized ligand (Scheme 1, FIG.2). Coupling a monoprotected diamine with the commercially availableDTPA anhydride, followed by deprotection provides a candidate ligand tobe coupled to the polymeric active ester. The net charge of the compoundonce complexed to lanthanide is −1. The facile synthesis of thischelator makes it an attractive starting point for optimizing thepolymeric backbone with attached chelators.

DTPA ligands are inherently more kinetically labile than the macrocyclicligand based on the cyclen framework. The macrocyclic nature of thecyclen-based ligands preorganizes the metal binding site, leading togreater thermodynamic and kinetic stability. These chelates are known tobe stable in vivo for days¹⁷. Reaction of commercially availabletritertbutylmethylcyclen (Macrocylics) with the readily availablehomoserine derivative provides an orthogonally protected DOTA derivative(Scheme 2, FIG. 2)¹⁸. The Fmoc protecting group can be removed to accessthe amine and make it available to couple with the polymeric backbone.In some instances it may be necessary to employ a spacer between theDOTA chelate and the polymer. A variety of selectively protected aminoacids of different lengths is commercially available and can be readilycoupled and deprotected to form linkers. The lanthanide complex of thischelate will carry a net −1 charge. Based on functionality, these Lnchelates with the reactive —NH₂ group are referred to as (L)-NH₂.

Polymer synthesis and chelate attachment: Herein below, the synthesis ofcandidate polymers, the attachment of functional chelates to the polymerbackbone, and the characterization of the metal containing polymers aredescribed. These are intended to be examples, and not to limit the scopeof the claims. Other examples can be used as is known to those skilledin the art.

Random copolymer poly(DMA-co-NAS): A recent report¹⁹ describes thesynthesis of a 75/25 mole ratio random copolymer (3, FIG. 3) ofN-acryloxysuccinimide (NAS) with N,N-dimethyl acrylamide (DMA) by RAFTwith high conversion, excellent molar mass control in the range of 5000to 130,000, and with Mw/Mn≈1.1. In this embodiment (Scheme 3, FIG. 3),the active NHS ester of 3, FIG. 3 is reacted with a liganded lanthanide(L) bearing a reactive amino group to yield the copolymer 4, FIG. 3.FIG. 15 is a flow chart showing the steps involved in RAFTpolymerization.

Poly(NMAS): Yet another approach has been reported by Müller²⁰ and usedto attach drug conjugates to the polymer backbone. In this approach,Müller polymerized NMAS by ATRP (Scheme 4, FIG. 3), obtaining polymerswith a mean molar mass ranging from 12 to 40 KDa with Mw/Mn ofapproximately 1.1. In their experiments, limiting amounts of variousdrugs or drug-mimics bearing a spacer and a primary amine were reactedwith the NHS ester groups of 5, FIG. 3, and then the remaining siteswere reacted with excess Me₂NH. Their initiator was the hydroxyethylester of bromoisobutyric acid; thus the polymer chains all had a primaryalcohol as an end group. Here samples of 5, FIG. 3, are reacted withexcess (L)-NH₂, maximizing the number of (L) groups that can be attachedto the polymer.

Poly(MAA): Another aspect of the Applicant's teaching is related tospecific functional advantages of polymer tags with a very narrow molarmass distribution. Polymethacrylic acid (PMAA) can be prepared byanionic polymerization of its t-butyl or trimethylsilyl (TMS) ester. Ifthe reaction is terminated with ethylene oxide prior to ester hydrolysis(FIG. 4), the polymer will bear a —CH₂CH₂—OH as a functional end group.A route for attaching (L) to the polymer involves reacting thetetrabutylammonium carboxylate salt of the polymer with thebromoacetamide derivative of (L)-NH₂ (Scheme 5, FIG. 4).

Poly(DMAEMA): Recently, samples of poly(dimethylaminoethyl methacrylate)(PDMAEMA) were prepared by ATRP²¹. This is a well-known polymer that isconveniently prepared with mean Mn values ranging from 2 to 35 KDa withMw/Mn of approximately 1.2 This polymer can also be synthesized byanionic polymerization with a narrower size distribution²². This polymercan be reacted with the bromoacetamide derivative of (L)-NH2. Thisyields a zwitterionic polymer 8, Scheme 6, FIG. 4, which has suitablewater solubility. The unreacted dimethylaminoethyl groups will beprotonated at neutral pH and contribute a small positive charge to thepolymer.

Spacers: A potential source of interference between a metal-bearingpolymer tag and affinity reagent activity is the close proximity of thebulky polymer when attached to the affinity reagent. Spacers, forexample, PEG spacers, can be situated between the linker and the polymeror between the polymer and the linker. Methods for the addition ofspacers is known to those skilled in the art.

The spacer can also be an integral part of the polymer backbone to helpmitigate this problem. In the applicant's teaching, the syntheses (forexample see Schemes 4-6, FIGS. 3 and 4) can be modified to create PEGblock copolymers. The PEG portion of the block copolymer serves as a PEGspacer, and the synthetic strategies make it possible to vary the PEGspacer length as needed in response to bioassay results that indicateproblems with binding efficiency or sensitivity. The spacer can be anyspacer as is known to those skilled in the art. For example, it can be aminimal spacer as shown in Scheme 12 and compound 12. This specificenactment seems to be novel as we are not aware of its priorapplication.

End-group control and coupling chemistry: According to the Chapmanreview on PEGylated antibodies¹³, approaches to PEG attachment viareaction with the free amino group of the lysine were successful, butthe PEGylated antibodies obtained exhibited reduced antigen bindingefficiency. It appears that the random nature of the chemical reactionto the various lysine groups in the antibody led to PEG attachment atsites that interfered with binding. A more benign result was obtainedfor the case in which the PEG chain was attached specifically to asingle cysteine in the FC fragment that was introduced into the antibodythrough site-specific mutation. Here reduction of a disulfide bondwithin the FC fragment of the antibody, followed by covalent attachmentof the polymers to one or both of the —SH groups formed is described.Thus a thiol reactive group may be used at one terminus of the polymers.

RAFT polymers: The thiobenzoate end group of RAFT polymers isconveniently converted to a terminal —SH group. This chemistry is shownin scheme 7a, FIG. 5, for polymer 4, FIG. 3. Numerous methods are known,to those skilled in the art, for crosslinking thiols, in analogy withreactions described for —SH terminated polyethylene glycol (PEG-SH)²³,and allow the attachment of the polymer via the mixed disulfide to thefree —SH of an antibody or other affinity reagent (denoted as“protein-SH”). Alternatively, bismaleimide derivatives are commerciallyavailable and alkylation of the polymer with these reagents followed byGPC (Gel Permeation Chromatography) purification and reaction with thefree thiol of the antibody or other affinity reagent provides thedesired conjugate²⁴.

ATRP polymers: Polymers of the structure 5, FIG. 3, reported by theMüller group²⁵ have a terminal —CH2CH2—OH group. A different initiatorfor the polymerization reaction is described here. 2,6-napthalenederivatives are readily available and will provide an orthogonallyprotected amine. After deprotection, reaction of the amine with abifunctional NHS-maleimide, the thiol-amine cross-linking agent willprovide the polymeric labeling agent for antibody conjugation. Thisinitiator also provides a convenient chromophore for quantification ofthe polymer. This also shown in scheme 7b in FIG. 5.

Anionic Polymerization (Scheme 5, FIG. 4): Anionic polymerizations canoften be terminated by reaction with functional electrophiles tointroduce an end group to the polymer²⁶. Enolates react effectively withallylic and benzylic halides²⁷. Quenching styrene polymerization withepichlorohydrin has been shown to be problematic²⁸. Conditions forquenching the enolate end of a living poly(t-butyl methacrylate) toyield the terminal epoxide are described here. While glycidylmethacrylate can be polymerized anionically at low temperature in thepresence of LiCI, which makes the propagating anion less nucleophilic²⁹,it is expected that the enolate of t-butyl methacrylate should ring-openan epoxy group at higher temperature³⁰. Opening of the epoxide withazide provides an orthogonal functional group stable under conditions ofester hydrolysis. Treatment of azides with an alkyne in the presence ofCu(I) salts yields triazoles in high yield³¹. By using this couplingreaction a thiol reactive maleimide is installed at the terminus of thepolymer. This is also shown in scheme 7c in FIG. 5.

Attachment (linker) groups: The attachment group provides a covalentbond between bioorganic (proteins, peptides, oligonucleotides)molecules, for example affinity reagents, and the element tag. Forexample, the linkage can be effected via thiols using a maleimidoattachment group; through the N-terminus or basic side chain (lysine,arginine, histidine) (see Scheme 8c, FIG. 6), through the C-terminus oracidic side chain (aspartic acid, glutamic acid) usingp-(chloromethyl)styrene (see Scheme 8c, FIG. 6), or via oxidation of thesugar moiety on the antibody or other affinity reagent and coupling viaa hydrazine group. One may take advantage of thiol groups created byreduction of the disulfide bond in the FC fragment of the antibody. Thiscombination “bioorganic molecule—attachment group—element tag” isthought to be described here for the first time.

Functional example of coupling chemistry: There are four main couplingchemistries commonly used to attach polymers (such as PEG) to the freethiols of proteins. The advantages and disadvantages of each of thesereactions have recently been reviewed³². One approach involves disulfideexchange as shown in Scheme 7a, FIG. 5. Three other common reactionsinvolve addition of —SH to a maleimide or a vinyl sulfone and thedisplacement of iodide from an iodoacetamide (Schemes 8a-c, FIG. 6). Toavoid the slow hydrolysis in water that is typical of maleimide andiodoacetamide groups, a strategy in which the thiol-reactive agent isadded to the end of the (L)-bearing polymer just prior to tagging of theaffinity reagent is possible. This strategy takes advantage of the“click” chemistry developed recently by Sharpless³³ (Scheme 8b, FIG. 6)involving the 1,3-dipolar addition of azides to acetylenes, a reactionthat Sharpless has shown to occur under mild conditions withquantitative yield. To introduce the acetylene unit on the end ofpolymers bearing a terminal —NH₂ group, they are reacted with an activeester derivative of 4-pentynoic acid. The polymer is then set up for areaction with a derivative of the form X—R—N3, where R is the spacer andX represents the thiol-reactive group.

Coupling of polymer to an antibody or other affinity reagent: As anexample, reduction of disulfide bonds in an antibody or other affinityreagent can be performed using immobilized trialkylphosphine TCEP(Tris[2-carboxyethyl] phosphine hydrochloride) covalently linked to abeaded agarose support (Pierce). TCEP is known to be an efficientreductant of alkyl disulfides over a wide range of pH and does notinterfere with commonly used sulfhydryl-reactive reagents such asmaleimide cross-linkers. The use of beads permits recovery of thereduced antibody or other affinity reagent by simple centrifugation fromthe reducing agent with subsequent separation from the beads.

Purification of polymer modified antibodies: Due to the large size ofthe IgG antibodies (150 KDa) one option is to separate the excessmetallated labeling polymer (20-40 KDa) from the antibody using gelfiltration chromatography. Alternatively, Protein A and Protein G havebeen used to purify antibodies.

As is known to those skilled in the art, the element or metal atoms canbe added to the polymer tag at different steps during the production ofthe tagged biomolecule. It is beneficial to add the element (metal) ofthe tag after conjugation of the antibody or other affinity reagent withthe ligand-polymer. This strategy has several advantages: i) conversionof antibody-ligand-polymer conjugate into antibody-metal-polymerconjugate can be done directly before bio-assay; ii) the multitude ofaffinity molecules can be tagged with the same ligand-polymer conjugateunder the same conditions. The choice of metal (or isotope) to use canbe determined directly before the multiplexed experiment by the reagentuser significantly increasing experimental flexibility; iii) decouplingof both tagging stages allows series of important independent controlexperiments in which the same antibody can be tagged with differentmetals; iv) selection of the internal standards is unhindered, and therelative sensitivity of the elemental analyzer can be effectivelycontrolled.

The order of steps for the synthesis of the tagged biomolecule can takemany forms. Three examples are provided below, but it is to beunderstood that other orders of steps are possible:

A B C Synthesize polymer Synthesize polymer Synthesize polymer Bindmetal to polymer Bind linker to polymer Bind linker to polymer Bindlinker to polymer Bind metal to polymer Bind linker to antibody Bindlinker to antibody Bind linker to antibody Bind metal to polymer

Further, the linker can be attached to the biomolecule before the linkeris attached to the polymer. Most often, the metals will be attachedanytime before binding the tagged affinity reagent to the analyte. It ispossible to add the metals after attaching the affinity reagent to theanalyte, but the background is expected to be elevated because manyanalytes, and in particular cells, will bind metals non-specifically. Itis therefore less likely to be performed successfully after binding theaffinity reagent to the analyte.

Further, the polymer element tag may be attached to a biomolecule whichis other than an affinity reagent. For example, the polymer element tagmay be attached directly to an analyte, for example but not limited to agrowth factor, cytokine or chemokine for studying kinetics ofligand-receptor interactions. Specifically, EGF (epidermal growthfactor) with polymer element tag may be used as a probe to investigateEGFR (epidermal growth factor receptor) abundance on cell surface,receptor dimerization and internalization. This aspect is also withinthe scope of the applicant's teachings. Two or more analytes may also beanalyzed in a multiplex reaction.

Aspects of the Applicant's teachings may be further understood in lightof the following examples, which should not be construed as limiting thescope of the present teachings in any way.

EXAMPLES Example 1. Synthesis of Copolymers of N,N-dimethylacrylamideand N-acryloxysuccinimide by RAFT Polymerization

N,N-dimethylacrylamide (DMA) and N-acryloxysuccinimide (NAS) werecopolymerized by the reversible addition-fragmentation chain transfer(RAFT) polymerization technique, to obtain random copolymer precursorswith side-groups statistically grafted via the reactive NAS units¹⁹. Therandom copolymers of DMA and NAS, poly(DMA-co-NAS), were prepared usingtert-butyl dithiobenzoate (t-BDB) as chain transfer agent (CTA) (Scheme9, FIG. 7).

Preparation of tert-Butyl Dithiobenzoate (t-BDB).³⁴ In a 500 mLround-bottomed flask equipped with a magnetic stirrer, 150 mL of adiethyl ether solution of s-(thiobenzoyl)thioglycolic acid (0.27 g, 2.4mmol) was added to 100 mL of an aqueous basic solution (NaOH, 1 mol L⁻¹)of sodium 2-methyl-2-propanethiolate (0.510 g, 2.9 mmol). This biphasicmixture was vigorously stirred at room temperature for 5 hours. Then,the purple ether phase was removed and washed twice with 500 mL of anaqueous basic solution (NaOH 1 mol L⁻¹) and twice with 500 mL of a 10%NaCl aqueous solution and dried over anhydrous magnesium sulfate.Purification by silica gel chromatography (Kiesegel-60) with petroleumether/ethyl acetate (99/1:v/v) as eluent gave tert-butyl dithiobenzoate(t-BDB) as a dark purple oil (90% yield). 1H NMR (CDCL3) d (ppm): 1.69(S, 9H, 3×CH3), 7.36 (m, 2H, meta-ArH), 7.50 (m, 1H, para-ArH) and 7.88(m, 2H, ortho-ArH).

Preparation of N-acryloxysuccinimide (NAS).³⁵ N-hydroxysuccinimide (10g, 0.086 mol) and triethylamine (13.2 g, 0.129 mol) were dissolved inchloroform (130 mL) at 0° C. Acryloyl chloride (8.6 g, 0.094 mol) wasadded dropwise over a period of 2 hours to the stirred reaction mixture.The reaction is described in Scheme 1, FIG. 2. After being stirred anadditional 30 minutes at 0° C., the solution was washed twice with 60 mLsaturated NaCl aqueous solution, dried over MgSO4, filtered andconcentrated so as to get a residual volume of 30 mL. An ethylacetate/pentane mixture (14 mL, 1:3 v/v) was added and the temperaturewas maintained at 0° C. to induce NAS crystallization overnight (70%yield). 1H NMR (CDCl3) d (ppm): 2.95 (S, 4H, CH2CH2), 6.20 (m, 1H,CH═CH2), 6.4 (m, 1H, CH═CH2) and 6.75 (m, 1H, CH═CH2).

Preparation of random copolymers of DMA and NAS. General experimentalconditions: DMA was distilled under reduced pressure prior to use.Monomers, t-BDB, initiator 2,2′-azobis(2-methylbutyronitrile) (AMBN) andsolvent dioxane were introduced in a schlenk tube equipped with amagnetic stirrer. The mixture was degassed by three freeze-vacuum-thawcycles and then heated under argon in a thermostated oil bath at 80° C.The percentage yields were calculated gravimetrically.

The structure of copolymers has been verified by application ofappropriate chromatographic and spectrometric methods. Gel permeationchromatography (GPC) has been used to establish the molecular weight andmolecular weight distribution of the copolymers. A Viscotek liquidchromatograph equipped with a Viscotek VE3210 UV/vis detector and aVE3580 reflective index detector and Viscotek GMHHR-M Viscogel™ GPCcolumn was used. The flow rate was maintained at 0.5 mL min-1 using aViscotek VE1121 GPC pump. 1-Methyl-2-pyrrolidinone was used as eluent.The molecular weights are provided as polystyrene equivalents. FIG. 15is a flow chart of the RAFT polymerization procedure.

Preparation of copolymer containing 13 mol % of NAS units. NAS (0.81 g,4.82 mmol), DMA (3.2 mL, 31 mmol), AMBN (70 mg, 0.36 mmol) and t-BDB(116 mg, 0.521 mmol) were added into 33 mL of 1,4-dioxane. The solutionin a schlenk tube was degassed and heated at 80° C. for 18 hours. Thenthe solution was cooled and precipitated in 300 mL diethyl ether. Thecollected solid was redissolved in 1,4-dioxane and precipitated indiethyl ether. Yield of dried polymer was 75%. The molecular weight andpolydispersity are shown in FIG. 8.

Preparation of copolymer containing 47 mol % of NAS units. NAS (2.33 g,13.9 mmol), DMA (N,N-dimethylacrylamide 1.6 mL, 15.5 mmol), AMBN (70 mg,0.36 mmol) and t-BDB (116 mg, 0.521 mmol) were added into 30 mL of1,4-dioxane. The solution in a schlenk tube was degassed and heated at80° C. for 18 hours. Some precipitation was observed in the tube. Thenthe solution was cooled and precipitated in 300 mL diethyl ether. Thecollected solid was redissolved in DMF and precipitated in diethylether. Yield of dried polymer was 80%. The molecular weight andpolydispersity are shown in FIG. 8.

Preparation of copolymer containing 60 mol % of NAS units. It wasprepared by a similar procedure as aforementioned (47 mol % of NASunits) one. More NAS monomer was added and solvent 1,4-dioxane wassubstituted by DMF. Yield of dried polymer was 80%. The molecular weightMn and polydispersity Mw/Mn are shown in FIG. 8.

Example 2. Preparation of Ligand-Polymer Conjugate

The following preparation of the polymer ligand conjugate is amenablefor use with any amine functionalized ligand according to Scheme 10 andScheme 11, FIG. 9.

To a stirred solution of the (N,N-dimethylacrylamide (DMA) andN-acryloxysuccinimide (NAS)) copolymer containing 47 mol % of NAS units(35 mg, 3.5461 mmol) and N,N-diisopropylethylamine (300 μl) in DMF/H2O(60:40, 1 mL) was added a solution of the amine pendant ligand 9, FIG. 9(78 mg) in the same mixture (2 ml). The reaction mixture was stirredovernight under nitrogen at room temperature. The solvent was removedunder vacuum and the solids were dissolved in H₂O. The solutions weredialyzed by repeated washings with deionized water (5×4 mL) in an Amiconcentrifugal filter (5K MW C.O.) The solution remaining in the filterdevice was concentrated to give a yellowish solid. The solid waspurified further by precipitation from methanol with diethylether togive a yellow powder (48 mg)

Ligand-polymer conjugate (5.5 mg) was dissolved in 1 mL of 50 mMphosphate buffer, (pH 8.5. 2 mL of 20 mM DTT) and the reaction mixturewas stirred for 1 hour at 50° C. After the reaction, the mixture wasmade acidic (pH 4) with acetic acid and washed in an Amicon centrifugalfilter (5K MW C.O.) with aqueous acetic acid (5 mM, 5×4 mL). Thesolution left in the filter device was then transferred to a smallreaction flask containing 2 mL of 100 mM phosphate buffer, pH 8.5. Asolution of 1,4-bis(maleimido)butane (50 equiv.; 32 mg) in DMF was addedto the flask and the reaction mixture was stirred overnight at 50° C.The solvent was evaporated to give a residue, which was dissolved inH₂O, and the clear solution was again washed using an Amicon centrifugalfilter (5K MW C.O.) with deionized water (5×5 mL). The supernatant waslyophilized to give the final conjugated polymer (4 mg).

Example 3. Preparation of Ligand-Polymer Conjugate: DOTA Based ConjugateAccording to Scheme 12, FIG. 10

To a stirred solution of the DMA-NAS copolymer with 60 mol % of NASunits (100 mg) in DMF (3 mL) and triethylamine (1 mL) was added asolution of amine pendant ligand 10 (363 mg, 0.590 mmol) in DMF (2 mL).The reaction mixture was stirred overnight under nitrogen at roomtemperature. After the solvent was removed under vacuum, the residue 11was dissolved in neat trifluoroacetic acid (3 mL) and stirred overnightat room temperature. The solution was evaporated, and the residue wastaken up in water and dialyzed by repeated washings with deionized water(6×5 ml) in an Amicon centrifugal filter (5K MW C. O.). The solutionremaining in the filter device (ca. 0.8 mL) was concentrated to give ayellow solid 12 (179 mg).

The entire sample of polymer-ligand conjugate 12 was dissolved in 50 mMphosphate buffer (pH 8.5, 2 mL) containing 20 mM DL-dithiothreitol, andthe reaction mixture was stirred for 1 hour at 50° C. After this time,the mixture was acidified to pH 4 with acetic acid, and washed in anAmicon centrifugal filter (5 K MW C. O.) with aqueous acetic acid (5 mM,5×5 mL). The solution left in the filter device (0.8 mL) was thentransferred to a small reaction flask containing phosphate buffer (100mM, pH 8.5, 5 mL). A solution of 2,2′-(Ethylenedioxy)bis(ethylmaleimide)(191 mg, 0.619 mmol) in DMF (2 mL) was added to the flask and thereaction mixture was stirred for 1 hour at room temperature. Water (3mL) was added into the flask and the solid was removed by filtration.The resulting clear solution was again washed with deionized water (5×5mL) using an Amicon centrifugal filter (5K MW C. O) and the supernatantwas purified by Sephadex G-50 Column with HPLC system using water as aneluent. The fraction was collected and lyophilized to give the finalconjugated polymer 13 (165.0 mg).

Example 4. Preparation of Ligand-Polymer Conjugate: DTPA Based ConjugateAccording to FIGS. 9, 11 and FIG. 16

To a stirred solution of the DMA-NAS copolymer with 60 mol % of NASunits (2.0 g) in DMF (30 mL) and triethylamine (4.3 mL) was added asolution of tert-butyl 2-aminoethylcarbamate, 14 (2.5 g, 15.6 mmol) inDMF (10 mL). The reaction mixture was stirred overnight under nitrogenat room temperature. Then the mixture was precipitated in 500 mL ofdiethyl ether. The collected solid 15 (400 mg) was dissolved in neattrifluoroacetic acid (3 mL) and stirred overnight at room temperature.The solution was evaporated, and the residue was taken up in water anddialyzed by repeated washings with deionized water (6×5 ml) in an Amiconcentrifugal filter (5K MW C. O.). The solution remaining in the filterdevice (ca. 0.8 mL) was concentrated to give a yellow solid 16 (210 mg).

DTPA succinimidic ester was prepared according to a publishedprocedure.³⁶ 16 g of DTPA (40.64 mmol) dissolved in 320 mL ofAcetonitrile (23 g, 230 mmol of triethylamine added). Solution wasstirred at 50° C. to dissolve the DTPA. 3.36 g ofdicyclohexylcarbodiimide (DCC, 16.3 mmol) and 1.9 g ofN-Hydroxysuccinimide (NHS, 16.3 mmol) were added simultaneously at roomtemperature. The reaction was carried out overnight. White precipitatewas observed and filtered off by filtration paper, generating solution(A).

210 mg of solid 16 (ca. 0.8 mmol amino groups) was dissolved in 80 mL ofdistilled water and added into solution (A) at room temperature. 5 mL oftriethylamine was added, and the solution was stirred at roomtemperature overnight. Solvents (triethylamine, acetonitrile) was thenevaporated and 100 mL water added. The solution was dialyzed (1K cut-offmembrane) for two days. Then the aqueous solution was concentrated, andacetic acid was added. It was dialyzed again with the same membrane foranother three days. The solution is concentrated to give a solid 17 (190mg).

Solid 17 (110 mg) was dissolved in 2.3 mL of phosphate buffer solution(pH 7.2). Then tri(2-carboxyethyl)phosphine (TCEP, 0.18 mL of 0.5 Msolution) was added into buffer solution at room temperature. After thesolution was stirred for 2 hours, it was added into2,2′-(ethylenedioxy)-bis(ethylmaleimide) (0.36 mmol, 106 mg) in 2.3 mLof DMF at room temperature. 100 mL of distilled water was added after 2hours and the solution was filtered through 5 k cut-off membrane with 5%DMSO/water (2 times) and then distilled water (3 times). The fractionwas collected and lyophilized to give the final conjugated polymer 18(90 mg). FIG. 16 is a flow chart showing the procedure forpolymer-DTPA-linker attachment procedures.

Example 5. Preparation of Ligand-Polymer Conjugate: Poly(MAA) orPoly(AA)

One aspect of the invention is related to specific functional advantagesof polymer tags with a very narrow molar mass distribution.Polymethacrylic acid [Poly(MAA)] or polyacrylic acid [Poly(AA)] can beprepared by anionic polymerization of its t-butyl or trimethylsilyl(TMS) ester.³⁷ If the reaction is terminated withtert-butyldimethylsilyl 3-chloropropyl sulfide,³⁸ prior to esterhydrolysis (see below), the polymer will bear a protected —SH functionalend group. They are reacted with tert-butyl 2-aminoethylcarbamate toform a polymer with protected amino groups, which is then hydrolyzedinto polymer 19 (FIG. 12, Scheme 13). The free amino groups on mainchain of polymer 19 offer sites for chelate attachment. The route forattaching chelate refers to the previous procedure using DTPAsuccinimidic ester (FIG. 11).

Poly(NMAS). Another approach has been reported by Müller³⁹ and used toattach drug conjugates to the polymer backbone. In this approach, NMASwas polymerized by ATRP, obtaining polymers with a mean molar massranging from 12 to 40 KDa with Mw/Mn of approximately 1.1. The initiatorused was the hydroxyethyl ester of bromoisobutyric acid; thus thepolymer chains all had a primary alcohol as an end group. Here, a newinitiator based on cystamine 20 can be prepared (FIG. 12, Scheme 14). Itis then used in the ATRP of NMAS to form a polymer 21 (FIG. 12, Scheme14) with disulfide group. The polymer 21 can be reacted with tert-butyl2-aminoethylcarbamate as shown in FIG. 12, Scheme 13 to generate apolymer with pendent amino groups for attachment of DTPA ligands. Byusing tri(2-carboxyethyl)phosphine (TCEP), the disulfide bond wasreduced and a thiol end-group was generated for attachment of a linkerto an antibody (FIG. 12, Scheme 15).

Example 6. Multiplex Labeling of Leukemia Cells

K562 cells, a model cell line of human chronic myeloid leukemia, werecultured under standard tissue culture conditions in DMEM (Dulbecco'sModified Eagle's Medium) supplemented with 10% FBS (fetal bovine serum),2 mM L-glutamine, and antibiotics. Growing cells were collected by lowspeed centrifugation (500×g), washed once with phosphate buffered saline(PBS), pH 7.4 and immunolabeled with primary antibodies attached to themetal-polymer conjugate (Ho, Dy, Nd, Eu, Pr, or La separately for eachantibody) as described in Scheme 10 and Scheme 11 (FIG. 9). Six cellsurface-specific antibodies were chosen for the experiment: CD38, CD110,CD61, CD45, CD54, CD49d. Aliquots of cells in triplicate tubes (0.3×106)were labeled with each antibody separately or with all antibodiescombined in one reaction mixture (sample ALL). As negative control,mouse IgG1 isotype immunoglobulins were attached to metal-polymerconjugates carrying the same elements as the primary antibodies—Ho, Dy,Nd, Eu, Pr, or La. After 30 minutes incubation on ice, the cells werewashed with PBS three times by centrifugation. The final cell pellet wasdissolved in concentrated HCl (35%), mixed with an equal volume of 1 ppbIr/HCl solution as internal standard and subjected to volume analysisICP-MS. Results are presented in FIG. 13.

Antibodies were attached to the metal-polymer conjugate (synthesizedaccording to Scheme 10 and Scheme 11, FIG. 9) according to the followingprotocol and reagents.

Reagents: Antibody at least 100-150 μg (˜1 nmol) in 100 μl PBS/EDTA (˜1mg/ml). The antibodies were purchased commercially from BD Biosciences,San Jose, Calif.).

TCEP disulfide reducing gel (4% cross-linked agarose beads) from Pierce#77712; supplied as 50% slurry. Used at 1:1 50% slurry to antibody v/v.

Ligand-Polymer Conjugate (see Scheme 11, FIG. 9) dissolved in doubledistilled water (ddH₂O). Expected MW 11,000.

R-Buffer is 0.1M sodium phosphate pH 7.2, 2.5 mM EDTA

C-Buffer is TBS, 1 mM EDTA

L-Buffer is 20 mM ammonium acetate pH 6.0

Reduction of IgG Disulfide Bonds: Added 200 μL R-Buffer and 50 μgantibody solution to Diafiltration Membrane.

Centrifuged 10,000 g for 10 minutes. Discarded flow-through. Repeatedonce.

Added 100 μL R-Buffer and 0.8 μL 0.5M TCEP solution to DiafiltrationMembrane and mixed gently (4 mM TCEP). Did not vortex.

Incubated 30 minutes at 37° C.

Added 200 μL C-Buffer. Centrifuged 10,000 g for 10 minutes. Discardedflow-through.

Labelling of Reduced IgG: Added 200 μL C-Buffer to membrane.

Prepared the element tag in C-Buffer at a concentration of 1 mM (1.1 mgelement tag in 50 μL C-Buffer).

Added 10 μL of the prepared element tag to the tube containing 200 μL ofthe reduced IgG solution and mixed well. Did not vortex.

Allowed the reaction to proceed at least 1 hour at 37° C.

Added 200 μL L-Buffer to Membrane. Centrifuged 10,000 g for 10 minutes.Discarded flow-through. Repeated twice.

Added 100 μL L-Buffer to membrane to resuspend labelled antibody.

Added 5 μL of 0.1M lanthanide solution (prepared in Ultrapure Water asis known to those skilled in the art) to the antibody conjugated withthe polymer tag. Mixed well. Did not vortex.

Incubated 30-60 minutes at 37° C.

Added 300 μL TBS. Centrifuged 10,000 g for 10 minutes. Discardedflow-through. Repeated three times.

Added 50 μL TBS. Gently pipetted several times to recover the conjugateand transfered to eppendorf tube.

Although ICP-MS was used in this analysis, it is to be understood thatother forms of elemental analysis could have been used and areencompassed in the scope of the applicant's teachings.

Further, although leukemia cells were targeted as the analyte it isunderstood that any cell or particle can be analyzed in a similarmanner.

Example 7. Analysis of Double Labeled Antibodies—Fluorescent Label andElement Tag

In this example, the double labeled antibodies facilitate presorting andsubsequent elemental analysis of rare cells in mixed samples byICP-MS-based flow cytometry.

In one instance demonstration of data congruence collected by flowcytometry (FACS) and ICP-MS of cells stained with dually labelledantibodies (CD33-FITC-Pr) was conducted.

Monoclonal antibodies against cell surface antigen CD33 conjugated tofluoresceneisothiocyonate (FITC) (CD33-FITC; GenTex Inc.) were taggedwith the polymer-DOTA-Pr construct. This dual labelled antibody willfurther be referred to as CD33-FITC-Pr. Several well characterized humanleukemia cell lines (KG1a, THP-1, Kasumi-3; ATCC Inc) were used in cellstaining studies. FACS analysis was performed on FACScalibur™ flowcytometer instrument (BD Biosciences Inc.) and ICP-MS data was obtainedusing ELAN DRCPlus (Perkin Elmer SCIEX). Live cells were washed by lowspeed centrifugation and incubated with CD33-FITC-Pr or CD33-FITC orCD33-Pr for antigen expression controls. Non-specific immunoglobulinbinding was monitored with mouse IgG-FITC, IgG-Pr or dual labelledIgG-FITC-Pr. Data presented in FIG. 14a shows that fluorescence obtainedfrom cells stained with dual labelled CD33-FITC-Pr are similar toCD33-FITC on all cell lines tested. Note that the KG1a cell line doesnot express CD33.

Likewise when CD33 expression was tested using element tagged antibodiesCD33-Pr and dual labeled CD33-FITC-Pr (FIG. 14b ), the normalizedresponses were similar.

Example 8. Particle Elemental Analysis Using a Mass Spectrometer BasedFlow Cytometer

The metal-polymer conjugate tags enable multiplexed assay in single cellformat to distinguish a rare (for example a diseased) cell in a complexsample (for example, blood). The method can be used to identify leukemiacells in a patient's blood sample by employing metal-polymer tagsconjugated to specific antibodies that recognize cell surface antigenspresent on the leukemia cells. For example, a positive multiplexstaining of some cells in the peripheral blood mononuclear sample withantibodies against CD33, CD34, CD38, CD13, CD15, CD36 (tagged withdifferent metals) and analyzed in a mass spectrometer based flowcytometer will indicate that the patient is developing acute monoblasticleukemia (AML-M5). In a similar manner, this method can be used toidentify and quantify other cells, or particles.

Example 9. Kits

The invention encompasses kits useful for the preparation for theelement tags and for carrying out the methods of the invention. The kitscan include at least one of the following items:

a polymer comprising at least one metal-binding pendant group whichcontains at least one metal atom or is capable of binding at least onemetal atom and further comprising a functional group that allows thepolymer to be attached to one of a linker, a spacer, or a biomolecule, ametal solution, reagents for the attachment of the linker, spacer orbiomolecule to the polymer, reagents for attachment of a functionalgroup to the linker or the spacer, reagents for attachment of a metal tothe polymer, affinity reagents including antibodies, buffers,instructions for preparing the element tag, instructions for attachingthe element tag to an affinity reagent, instructions for attaching ametal to the element tag. and instructions for using the element tagsfor the analysis of analytes by elemental analysis. For example, thepolymer can be homopolymers or copolymers of acrylamides,methacrylamides, acrylate esters, methacrylate esters, acrylic acid andmethacrylic acid. The reagents can be chosen from at least one of thefollowing: TCEP (tri(2-carboxyethyl)phosphine),Ligand-Polymer-Linker-Spacer Conjugate, phosphate buffer, TBS(tris-buffered saline), EDTA (Diaminoethanetetraacetic acid), ammoniumacetate buffer, antibodies, metal salt solution, lanthanide saltsolution, blocker buffers, washing buffers, FBS (fetal bovine serum),DMEM (Dulbecco's Modified Eagle's Medium), BSA (bovine serum albumin),dithiothreitol, bismaleimide, and DMF (dimethylformamide). The polymercan be provided which is attached to a linker or attached to both alinker and a spacer.

All references cited are incorporated by reference.

While the applicant's teachings are described in conjunction withvarious embodiments, it is not intended that the applicant's teachingsbe limited to such embodiments. On the contrary, the applicant'steachings encompass various alternatives, modifications, andequivalents, as will be appreciated by those of skill in the art.

REFERENCE LIST

-   1. Baranov, V. I.; Bandura, D. R.; Tanner, S. D. European Winter    Conference on Plasma Spectrochemistry, Hafjell, Norway 2001, Book of    Abstracts, p. 85.-   2. Baranov, V. I.; Quinn, Z.; Bandura, D. R.; Tanner, S. D. Anal.    Chem. 2002, 74, 1629-36.-   3. Baranov, V. I.; Quinn, Z. A.; Bandura, D. R.; Tanner, S. D. J.    Anal. Atom. Spectrom. 2002, 17, 1148-52.-   4. Quinn, Z. A.; Baranov, V. I.; Tanner, S. D.; Wrana, J. L. J.    Anal. Atom. Spectrom. 2002, 17, 892-96.-   5. Baranov, V., Tanner, S., Bandura, D., and Quinn, Z. Kit for    detecting/measuring transition element, comprising tag having    transition element for tagging biologically active material and    instruction for tagging material, combining tagged material with    analyte, detecting/measuring elements. MDS, S. C. I. E. and MDS INC.    [US2004072250-A1; WO2005003767-A2].-   6. Baranov, V., Tanner, S., Bandura, D., and Quinn, Z. Detecting and    measuring transition elements e.g. isotope or ions, in a sample,    comprises tagging biologically active materials, and detecting and    measuring reactant complexes by an atomic mass or optical    spectrometer. MDS, S. C. I. E. and MDS INC. [WO200254075-A;    EP1348127-A; US2002086441-A1; WO200254075-A1; EP1348127-A1;    AU2002215784-A1; JP2004516490-W].-   7. Baranov, V. I.; Quinn, Z.; Bandura, D. R.; Tanner, S. D. Anal.    Chem. 2002, 74, 1629-36.-   8. Bandura, D. R., Baranov, V., I, Tanner, S., and Tanner, S. D.    Elemental flow cytometer, e.g. mass spectrometer or optical emission    spectrometer based cytometer used in, e.g. health science, food    sciences, environmental sciences, and genomics and proteomics, has    spectrometer. Bandura, D. R., Baranov, V., I, Tanner, S., and MDS    INC. [US2005218319-A1; WO2005093784-A1].-   9. Hsu, K. C.; Zabriskie, J. B.; Rifkind, R. A. Science 1963, 142,    1471-&.-   10. Powell, R. D.; Halsey, C. M. R.; Spector, D. L.; Kaurin, S. L.;    McCann, J.; Hainfeld, J. F. Journal of Histochemistry &    Cytochemistry 1997, 45, 947-56.-   11. Riddle, S. M.; Vedvik, K. L.; Hanson, G. T.; Vogel, K. W.    Analytical Biochemistry 2006, 356, 108-16.-   12. Shunmugam, R.; Tew, G. N. J. Am. Chem. Soc. 2005, 127, 13567-72.-   13. Chapman, A. P. Advanced Drug Delivery Reviews 2002, 54, 531-45.-   14. Guddat, L. W.; Herron, J. N.; Edmundson, A. B. Proc. Natl. Acad.    Sci. U.S.A 1993, 90, 4271-75.-   15. Parker, D.; Dickins, R. S.; Puschmann, H.; Crossland, C.;    Howard, J. A. Chem. Rev. 2002, 102, 1977-2010.-   16. Liu, S.; Edwards, D. S. Bioconjug. Chem. 2001, 12, 7-34.-   17. Caravan, P.; Ellison, J. J.; McMurry, T. J.; Lauffer, R. B.    Chem. Rev. 1999, 99, 2293-352.-   18. Baldwin, J. E.; North, M.; Flinn, A. Tetrahedron 1988, 44,    637-42.-   19. Relogio, P.; Charreyre, M. T. C.; Farinha, J. S. P.;    Martinho, J. M. G.; Pichot, C. Polymer 2004, 45, 8639-49.-   20. Godwin, A.; Hartenstein, M.; Muller, A. H.; Brocchini, S. Angew.    Chem. Int. Ed Engl. 2001, 40, 594-97.-   21. Wang, X. S.; Dykstra, T. E.; Salvador, M. R.; Manners, I.;    Scholes, G. D.; Winnik, M. A. J. Am. Chem. Soc. 2004, 126, 7784-85.-   22. Shen, Y.; Zeng, F.; Zhu, S.; Pelton, R. Macromolecules 2001, 34,    144-50.-   23. Woghiren, C.; Sharma, B.; Stein, S. Bioconjug. Chem. 1993, 4,    314-18.-   24. Green, N. S.; Reisler, E.; Houk, K. N. Protein Sci. 2001, 10,    1293-304.-   25. Godwin, A.; Hartenstein, M.; Muller, A. H.; Brocchini, S. Angew.    Chem. Int. Ed Engl. 2001, 40, 594-97.-   26. Hirao, A.; Hayashi, M. Acta Polymerica 1999, 50, 219-31.-   27. Mizawa, T.; Takenaka, K.; Shiomi, T. Journal of Polymer Science    Part A: Polymer Chemistry 2000, 38, 237-46.-   28. Xie, H. Q.; Pan, S. B.; Guo, J. S. European Polymer Journal    2003, 39, 715-24.-   29. Hild, G.; Lamps, J. P.; Rempp, P. Polymer 1993, 34, 2875-82.-   30. Takenaka, K.; Hirao, A.; Nakahama, S. Polymer International    1995, 37, 291-95.-   31. Cozzi, P. G.; Hilgraf, R.; Zimmermann, N. European Journal of    Organic Chemistry 2004.-   32. Roberts, M. J.; Bentley, M. D.; Harris, J. M. Adv. Drug Deliv.    Rev. 2002, 54, 459-76.-   33. Wu, P.; Feldman, A. K.; Nugent, A. K.; Hawker, C. J.; Scheel,    A.; Voit, B.; Pyun, J.; Frechet, J. M.; Sharpless, K. B.;    Fokin, V. V. Angew. Chem. Int. Ed Engl. 2004, 43, 3928-32.-   34. Favier, A.; Charreyre, M. T.; Chaumont, P.; Pichot, C.    Macromolecules 2002, 35, 8271-80.-   35. D'Agosto, F.; Charreyre, M. T.; Pichot, C. Macromolecular    Bioscience 2001, 1, 322-28.-   36. Rebizak, R.; Schaefe, M.; Dellacherie, E. Bioconjug. Chem. 1997,    8, 605-10.-   37. Mori, H.; Muller, A. H. Prog. Polym. Sci 2003, 28, 1403-39.-   38. Tohyama, M.; Hirao, A.; Nakahama, S.; Takenaka, K.    Macromolecular Chemistry and Physics 1996, 197, 3135-48.-   39. Godwin, A.; Hartenstein, M.; Muller, A. H.; Brocchini, S. Angew.    Chem. Int. Ed Engl. 2001, 40, 594-97.

What is claimed is:
 1. A kit comprising: an isotopic compositioncomprising multiple lanthanide atoms of a single isotope of alanthanide; and an element tag comprising a linear or branched polymercomprising a plurality of chelating groups, wherein each chelating groupof the element tag includes at least one lanthanide atom of the isotopiccomposition or is capable of binding at least one lanthanide atom of theisotopic composition; wherein: the isotopic composition does notcomprise a natural mixture of isotopes, and the kit does not compriseany radioactive lanthanide.
 2. The kit of claim 1, wherein the elementtag is functionalized to bind a biomolecule.
 3. The kit of claim 1,wherein the element tag is covalently attached to a biomolecule.
 4. Thekit of claim 1, wherein the kit further comprises a biomolecule.
 5. Thekit of claim 4, wherein the biomolecule is an oligonucleotide.
 6. Thekit of claim 4, wherein the biomolecule is an antibody.
 7. The kit ofclaim 1, wherein each chelating group includes at least one lanthanideatom of the isotopic composition.
 8. The kit of claim 1, wherein theisotopic composition is a lanthanide solution provided separate from theelement tag, and wherein each chelating group of the element tag iscapable of binding at least one lanthanide atom of the isotopiccomposition.
 9. The kit of claim 1, further comprising an additionalisotopic composition, wherein the additional isotopic compositioncomprises multiple additional lanthanide atoms of an additional singleisotope of a lanthanide that is different from the single isotope of thelanthanide of the isotopic composition.
 10. The kit of claim 9, furthercomprising an additional element tag comprising an additional linear orbranched polymer comprising a plurality of additional chelating groups.11. The kit of claim 10, wherein each chelating group of the linear orbranched polymer of the element tag includes at least one lanthanideatom of the isotopic composition, and wherein each additional chelatinggroup of the additional linear or branched polymer of the additionalelement tag includes at least one additional lanthanide atom of theadditional isotopic composition.
 12. The kit of claim 11, wherein eachelement tag is covalently bound to a different antibody.
 13. The kit ofclaim 1, wherein each chelating group is capable of binding at least onelanthanide atom of the isotopic composition, and each chelating group isselected from the group consisting of Diethylenetriaminepentaacetate(DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA),a DTPAderivative, and a DOTA derivative.
 14. The kit of claim 1, whereineach chelating group has a negative charge, when bound to a lanthanideatom.
 15. The kit of claim 1, further comprising a reagent for covalentattachment of the element tag to an antibody.